Vitreoscilla血紅素與D型胺基酸氧化酵素融合蛋白之催化特性

CATALYTIC PROPERTIES OF THE FUSION PROTEIN OF VITREOSCILLA HEMOGLOBIN WITH D-AMINO ACID OXIDASE

王鴻文(Hong-wen Wang)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

D-amino acid oxidase (DAO)為一種flavoprotein，可以轉化D型胺基酸成對應α-酮酸，並消耗氧氣，產生氨氣和過氧化氫。Vitreoscilla hemoglobin (VHb)是Vitreoscilla sp.在氧氣不足的環境下合成的細菌性血紅素，可提升菌體的生長與增加蛋白質和代謝物的生成。由於VHb對氧分子具有高吸附及脫附的特性，而DAO需要氧氣參與催化反應，故以分子生物技術將此二酵素融合後，檢視VHb對DAO催化作用的影響。將VHb與DAO的cDNA基因融合後，送入E. coli strain BL-21(DE3)異源表現，產生融合蛋白VHb-DAO。以TB進行培養時，於37ºC下以IPTG誘導8小時後，可測得最高DAO活性約為4.44 U/ml；但當誘導溫度下降至30ºC時，DAO活性表現高峰雖延遲至誘導後10小時，但可測得最高DAO活性約7.1 U/ml。未融合Trigonopsis variabilis DAO和 VHb-DAO有相同的最適反應pH 8.5和最適反應溫度50ºC，但熱與pH穩定性仍以未融合T. variabilis DAO較佳。於55ºC下處理30 min，前者仍有67%活性殘存，而融合蛋白僅39%活性殘存。在pH 9.0下處理30 min後，前者仍有75%活性殘存，而後者僅約57%活性殘存。而D-alanine 對熱穩定度的改善，以未融合T. variabilis DAO較佳；但對pH穩定度的改善，則兩者近似。

關鍵字

融合蛋白 ； Vitreoscilla血紅素 ； D型胺基酸氧化酵素

並列摘要

D-amino acid oxidase (DAO) is a flavoprotein which can catalyze the oxidative deamination of D-amino acids to α-keto acids, along with oxygen consumption and generation of ammonia and hydrogen peroxide. Vitreoscilla hemoglobin (VHb) is synthesized by Vitreoscilla sp. under oxygen-poor condition to improve cell growth as well as the synthesis of proteins and metabolites. Since VHb owes high rate constants for oxygen binding and release and DAO requires oxygen for catalysis, the effect of VHb on the catalytic activity of VHb-DAO fusion protein was examined. The VHb and DAO cDNA genes were fused and heterologously expressed in E. coli strain BL-21(DE3). The maximal activity of the fusion protein expressed in TB medium was 4.44 U/ml, which was obtained 8 hours after induction with IPTG at 37ºC. When the induction temperature was lowered to 30ºC, the maximal DAO activity expressed was 7.1 U/ml, which was obtained 10 hours after induction. The optimal pH and temperature for catalysis of unfused T. variabilis DAO and VHb-DAOTv were similar, both at 8.5 and 50ºC. However, the former had better thermal and pH stability. When heat treated at 55℃for 30 min, they had 67 and 39% residual activity left, respectively. When treated in pH 9.0 for 30 min, the former had 75% activity remained and the latter had only 57% activity left. The presence of D-alanine could improve both thermal and pH stability of the fusion protein. The improvement in thermal stability of unfused T. variabilis was more obvious than that of VHb-DAOTv, whereas its effect on pH stability were similar for both proteins.

並列關鍵字

fusion protein ； Vitreoscilla hemoglobin ； D-amino acid oxidase

參考文獻

Alonso, J., Barredo, J. L., Díze, B., Mellado, E., Salto, F., García, J. L. and Cortés, E. 1998. D-amino acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology. 144：1095-1101.

Alonso, J., Barredo, J. L., Armisén, P., Díze, B., Salto, F., Guisán, J. M., García, J. L. and Cortés, E. 1999. Engineering the D-amino acid oxidase from Trigonopsis variabilis to facilitate its overproduction in Escherichia coli and its downstream processing by tailor-made matal chelate supports. Enzyme Microb. Technol. 25：88-95.

Bailey, J. E., Lilius, G., Holmberg, N. and Bülow, L. 1999. The metabolic effects of native and transgenic hemoglobins on plants. Tibtech. January. 17：21-24.

Bailey, J. E.and Khosla, C. 1988. The Vitreoscilla hemoglobin gene：molecular cloning, nucleotide sequence and genetic expression in Escherichia coli. Mol. Gen. Genet. 214：158-161.

Bailey, J. E., Kallio, P. T. and Tsai, P. S. 1996. Expression of Vitreoscilla hemoglobin is superior to horse heart myoglobin or yeast flavohemoglobin expression for enhancing Escherichia coli growth in a microaerobic bioreactor . Biotechnol. Prog. 12：751-757.